Biology Reference
In-Depth Information
droplets and for quantification of intracellular lipid content in various biological
systems, such as mammalian cells (
Fowler & Greenspan, 1985
), ciliates (
Cole,
Fok, Ueno, & Allen, 1990
), bacteria (
Spiekermann, Rehm, Kalscheuer,
Baumeister, & Steinb¨chel, 1999
), yeast (
Kimura, Yamaoka, & Kamisaka,
2004
), fungus (
Kamisaka, Noda, Sakai, & Kawasaki, 1999
), and microalgae such
as diatom (
McGinnis, Dempster, & Sommerfeld, 1997
),
Botryococcus
(
Lee, Yoon,
& Oh, 1998
), haptophyte (
Eltgroth, Watwood, & Wolfe, 2005
),
Scenedesmus
and
Neochloris
(
da Silva, Reis, Medeiros, Oliveira, & Gouveia, 2009
);
Nannochloropsis
(
Elsey, Jameson, Raleigh, & Cooney, 2007
),
Chlorella
(
Chen, Zhang, Song,
Sommerfeld, & Hu, 2009
), and
Chlamydomonas
(
Moellering & Benning, 2010
).
However, staining protocols using this dye, especially with microalgae, vary greatly
and indeed the accuracy of Nile red staining method might be affected by many
factors, such as cell type, cell concentration, dye concentration, staining duration,
staining temperature, wavelengths for excitation and emission. The thick and rigid
cell walls associated with some microalgae, like
Scenedesmus dimorphus
, may pre-
vent the Nile red dye from effectively penetrating the cell wall and cytoplasmic
membrane and the subsequent binding with the intracellular neutral lipid. In this
situation, a modified protocol with staining at an elevated temperature, or even
assisted by a microwave and/or using the solvent dimethyl sulfoxide as the stain
carrier was developed (
Chen, Sommerfeld, & Hu, 2011; Chen et al., 2009
). The
Nile red staining conditions for microalgae have recently been optimized by
Cirulis, Strasser, Scott, and Ross (2012)
.
Besides being employed as a neutral lipid probe for visualizing oil droplets and
quantifying neutral lipid content, Nile red is widely used in high-throughput forward
genetic screens for lipid mutants in
Chlamydomonas
. This is made possible because
plasmid-mediated insertional mutagenesis has become one of the most powerful
tools in forward genetic studies aimed at identifying genes in a given process in
Chla-
mydomonas
(
Dent, Haglund, Chin, Kobayashi, & Niyogi, 2005
). We use the cell
wall-less strain
dw15-1
for insertional mutagenesis with plasmid pHyg3 carrying
hygromycin B resistance as the positive selection marker (
Berthold, Schmitt, &
Mages, 2002
). Nuclear transformation is achieved by the glass bead method as de-
scribed (
Kindle, 1998
) except that the plasmid is linearized with
Nde
I prior to trans-
formation and transformed cells are suspended in 0.5 mL Tris-acetate-phosphate
(TAP) medium and plated onto 2% agar supplemented with hygromycin. Using this
method, we have routinely produced several thousand independent hygromycin-
resistant transformants per experiment and transformation efficiency is no longer
limiting for mutant isolation. To screen for mutants defective in oil accumulation,
we grow transformed cells in mediumwith limited nitrogen (1 mM) in 96-well plates
to induce oil biosynthesis. After 4 days of growth, an appropriate amount of cells are
transferred to a new 96-well plate. Specific fluorescence is recorded using a plate
reader with fluorescence spectrometric capability before and after the addition of
Nile red. This first round of measurement may yield gain-of-function mutants that
produce oil under noninducing conditions or mutants that overproduce oil due to de-
fects in oil breakdown. Nile red specific fluorescence ratios are quantified again after
